Applications of Infrared and Raman Spectroscopies to Probiotic Investigation

In this review, we overview the most important contributions of vibrational spectroscopy based techniques in the study of probiotics and lactic acid bacteria. First, we briefly introduce the fundamentals of these techniques, together with the main multivariate analytical tools used for spectral interpretation. Then, four main groups of applications are reported: (a) bacterial taxonomy (Subsection 4.1); (b) bacterial preservation (Subsection 4.2); (c) monitoring processes involving lactic acid bacteria and probiotics (Subsection 4.3); (d) imaging-based applications (Subsection 4.4). A final conclusion, underlying the potentialities of these techniques, is presented

Applications of Infrared and Raman Spectroscopies to Probiotic Investigation

In this review, we overview the most important contributions of vibrational spectroscopy based techniques in the study of probiotics and lactic acid bacteria. First, we briefly introduce the fundamentals of these techniques, together with the main multivariate analytical tools used for spectral interpretation. Then, four main groups of applications are reported: (a) bacterial taxonomy (Subsection 4.1); (b) bacterial preservation (Subsection 4.2); (c) monitoring processes involving lactic acid bacteria and probiotics (Subsection 4.3); (d) imaging-based applications (Subsection 4.4). A final conclusion, underlying the potentialities of these techniques, is presented.Facultad de Ciencias Exacta

Detection of Microorganisms using MALDI and ion mobility mass spectrometry

Matrix-assisted laser ablation desorption ionization MALDI and ion mobility (IM) MALDI mass spectrometry (MS) were used for the detection and identification of microorganisms. MALDI MS is an analytical tool that separates ions by their mass-to-charge ratio (m/z) and is routinely used for bioanalysis because of its sensitivity, selectivity, general applicability, and tolerance to impurities. Ion mobility is a gas phase technique that separates ions based on their charge and collision cross-section. In this research, MALDI-TOF MS and MALDI-IM-TOF MS analysis were conducted in parallel to assess the effectiveness of MALDI-IM-TOF MS for microorganism identification. Whole cell bacteria Escherichia coli strain W 9637 and Bacillus subtilis 6633 were prepared and analyzed using both MALDI-TOF MS and MALDI-IM-TOF MS. The signals from both analysis methods were identified using a microbial database. Vacuum ultraviolet (VUV) post-ionization MALDI-IM-TOF MS was also used and additional peaks that could not be detected using MALDI-TOF MS and MALDI-IM-TOF MS were observed from B. subtilis. MALDI MS was used in combination with mass spectral fingerprinting software for the identification of whole cell bacteria in the presence of potential environmental interferants. Whole bacteria were analyzed in the presence of fumed silica, bentonite, and pollen from Juglans nigra (black walnut) at various mass ratios. The effect of the interferants on the identifications of bacteria at the genus and species level was evaluated using the bacteria fingerprinting software MB. The results showed that correct species identification for E. coli 35218, could be determined with fumed silica, bentonite, and pollen at a mass ratio of 1:1; whereas, at the same mass ratio, with diesel particulate, only genus identification could be made. Species identification for E. aerogenes 13048 with fumed silica and pollen at a mass ratio of 1:1 was achieved. Genus identification was determined for E.aerogenes with bentonite and diesel particulate. As the mass ratio of the interferant increased, the likelihood of species identification decreased with the exception of E. aerogenes with fumed silica and pollen. Under ambient conditions, laser ablation sample transfer using a mid-infrared laser at 2.94 ƒÝm was used to ablate gram-negative E. coli 35218 and gram-positive B. cereus 11178 bacterial colony particulate from a petri dish into a solvent droplet suspended above the petri dish. The solvent droplet containing the captured material was then transferred to a nanostructured-assisted laser desorption ionization (NALDI) target for analysis on a matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometer (MS). Several peaks that were observed in the NALDI spectra of both gram-negative and gram-positive correspond to phospholipid classes, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Additional phospholipids diglycosyldiglyceride (DGDG), triacylglyceride (TAG) and a lipopeptide, which are typically found in gram-positive bacteria were observed in the NALDI spectrum of B. cereus. Using LAST NALDI, phospholipids could be identified from both bacterial species without any sample pretreatment

Applications of Infrared and Raman Spectroscopies to Probiotic Investigation

In this review, we overview the most important contributions of vibrational spectroscopy based techniques in the study of probiotics and lactic acid bacteria. First, we briefly introduce the fundamentals of these techniques, together with the main multivariate analytical tools used for spectral interpretation. Then, four main groups of applications are reported: (a) bacterial taxonomy (Subsection 4.1); (b) bacterial preservation (Subsection 4.2); (c) monitoring processes involving lactic acid bacteria and probiotics (Subsection 4.3); (d) imaging-based applications (Subsection 4.4). A final conclusion, underlying the potentialities of these techniques, is presented.Facultad de Ciencias Exacta

Use of Fourier Transform Infrared Spectroscopy for the Classification and Identification of Bacteria of Importance to the Food Industry

The aim of this work was to use Fourier Transform Infrared Spectroscopy to characterize and identify bacteria of particular significance to the food industry. FT-IR spectroscopy is a rapid technique that can be applied to all groups of bacteria. The two objectives were to determine a suitable sampling procedure to record a spectrum and to determine a suitable statistical technique to identify characteristic regions of the spectrum associated with the genus and, potentially, the species. Pure cultures of bacteria were grown in broth, suspended in saline and dried to produce a film on a halide salt crystal. These films were then used to produce FT-IR spectra. In total, 80 spectra were recorded from seven genera, seven species and four strains of bacteria. Some of the spectra were considered to be too low in intensity to be included in statistical analysis. Data points from three specific windows of the remaining spectra were used to determine spectral distances between spectra. These spectral distances were used to perform cluster analysis using Ward\u27s method, the Complete Linkage method and the Centroid method. The statistical analysis created successful clusters for several of the species used but was inconclusive overall in being able to distinguish between spectra at the genus, species and strain level. This may be due to inconsistent growth of bacteria and insufficient manipulation of the data. This study has shown the potential for FT-IR spectroscopy to be used to identify bacteria with significance for food but further development is needed to reproduce the consistent results demonstrated in current literature

Novel technologies: study for application to fresh dairy sector

Ricotta is a potential growth medium for a wide range of microorganisms and it is an easily perishable food, characterized by a short shelf life (2-3 days), even under refrigerated conditions. Heat-based treatments can provoke microbial reduction, but alteration of the chemical and sensory profile can also occur. Therefore, in this Ph-D research alternative non-thermal technologies were tested on artisanal and industrial ricotta cheese to optimize parameters that promote quality preservation. The technologies tested were summarized in the figure 1 reported below (X-rays, Ultraviolet (UV-C) and Near Ultraviolet visible light (NUVL), Pulsed Light (PL) and Cold plasma). Specifically, artisanal and/or industrial ricotta were adopted for testing the effects of the above-mentioned techniques. In the first test artisanal and industrial ricotta samples were irradiated at 0.5, 2.0 and 3.0 kGy. At the two highest intensities (2.0 and 3.0 kGy), the artisanal product remained acceptable for more than 20 days, while the untreated samples became unacceptable after only 3 days of storage. The shelf life of the product irradiated at 0.5 kGy was limited to 14 days, due to the appearance of sensory defects. The industrial product irradiated at all X-rays intensities recorded a significant extension of the shelf life up to 84 days compared to the control which was discarded after 40 days due to sensory defects. As regard PL treatments at increasing fluence (1.3, 3.1, 7.5, 15.0 J/cm2) it was observed that PL at 1.3 and 3.1 J/cm2 allowed to delay microbial spoilage during storage, but higher fluences favored microbial growth and odor formation, possibly due to the surface nature of the PL technology. The analyses carried out on the product demonstrated that PL induced the formation of small protein particles, capable of interacting with lipids and carbohydrates and reorganizing into larger aggregates. Aggregation reduced protein solubility and occurred after exposure to hydrophobic protein groups. The photoreaction of proteins was confirmed by the formation of melanoidins and 8 carbonyls. Ricotta cheese artificially inoculated with Pseudomonas fluorescens was also tested by UV-C and NUVL. Results highlighted that the control samples became unacceptable after less than 5 days, while treated samples remained acceptable for more than 6 days. Finally, two different plasma-assisted approaches were assessed to extend the shelf life of artisanal ricotta cheese. In one test the plasma is applied indirectly, with plasma ignited in a controlled atmosphere (91% of N2 plus 9% of O2 supply gas) and the gaseous effluents (no plasma) directed to flow through small sterile tubes containing the cheese. In the other test product was directly exposed to the effects of the plasma. Treated and untreated samples were stored at 4 °C for a period of 8 days, during which microbiological, sensory and pH measurements were carried out. The results of the two approaches were similar: the concentrations of viable cells in the treated samples remained lower than those recorded in the control cheese, thus making the product more stable. From a sensorial point of view, the properties of ricotta in treated cheeses have been better preserved. Therefore, also cold plasma was effective in prolonging ricotta cheese shelf life

Desenvolvimento de espectroscopia de infravermelho para avaliar a qualidade bacteriana em alimentos

Doutoramento em BiologiaRapid and specific detection of foodborne bacteria that can cause food spoilage or illness associated to its consumption is an increasingly important task in food industry. Bacterial detection, identification, and classification are generally performed using traditional methods based on biochemical or serological tests and the molecular methods based on DNA or RNA fingerprints. However, these methodologies are expensive, time consuming and laborious. Infrared spectroscopy is a reliable, rapid, and economic technique which could be explored as a tool for bacterial analysis in the food industry. In this thesis it was evaluated the potential of IR spectroscopy to study the bacterial quality of foods. In Chapter 2, it was developed a calibration model that successfully allowed to predict the bacterial concentration of naturally contaminated cooked ham samples kept at refrigeration temperature during 8 days. In this part, it was developed the methodology that allowed the best reproducibility of spectra from bacteria colonies with minimal sample preparation, which was used in the subsequent work. Several attempts trying different resolutions and number of scans in the IR were made. A spectral resolution of 4 cm-1, with 32 scans were the settings that allowed the best results. Subsequently, in Chapter 3, it was made an attempt to identify 22 different foodborne bacterial genera/species using IR spectroscopy coupled with multivariate analysis. The principal component analysis, used as an exploratory technique, allowed to form distinct groups, each one corresponding to a different genus, in most of the cases. Then, a hierarchical cluster analysis was performed to further analyse the group formation and the possibility of distinction between species of the same bacterial genus. It was observed that IR spectroscopy not only is suitable to the distinction of the different genera, but also to differentiate species of the same genus, with the simultaneous use of principal component analysis and cluster analysis techniques. The utilization of IR spectroscopy and multivariate statistical analysis were also investigated in Chapter 4, in order to confirm the presence of Listeria monocytogenes and Salmonella spp. isolated from contaminated foods, after growth in selective medium. This would allow to substitute the traditional biochemical and serological methods that are used to confirm these pathogens and that delay the obtainment of the results up to 2 days. The obtained results allowed the distinction of 3 different Listeria species and the distinction of Salmonella spp. from other bacteria that can be mistaken with them. Finally, in chapter 5, high pressure processing, an emerging methodology that permits to produce microbiologically safe foods and extend their shelf-life, was applied to 12 foodborne bacteria to determine their resistance and the effects of pressure in cells. A treatment of 300 MPa, during 15 minutes at room temperature was applied. Gram-negative bacteria were inactivated to undetectable levels and Gram-positive showed different resistances. Bacillus cereus and Staphylococcus aureus decreased only 2 logs and Listeria innocua decreased about 5 logs. IR spectroscopy was performed in bacterial colonies before and after HPP in order to investigate the alterations of the cellular compounds. It was found that high pressure alters bands assigned to some cellular components as proteins, lipids, oligopolysaccharides, phosphate groups from the cell wall and nucleic acids, suggesting disruption of the cell envelopes. In this work, bacterial quantification and classification, as well as assessment of cellular compounds modification with high pressure processing were successfully performed. Taking this into account, it was showed that IR spectroscopy is a very promising technique to analyse bacteria in a simple and inexpensive manner.A deteção rápida e específica de bactérias que podem provocar deterioração de alimentos ou doenças associadas ao seu consumo é cada vez mais importante na indústria alimentar. A deteção, identificação e classificação de bactérias são geralmente realizadas utilizando métodos tradicionais baseados em testes bioquímicos e/ou serológicos e em métodos moleculares baseados em análise de DNA ou RNA. Contudo, estas metodologias são dispendiosas, demoradas e trabalhosas. A espectroscopia de infravermelho é uma técnica confiável, rápida e económica, que pode ser explorada como ferramenta para a indústria alimentar. Nesta tese foi avaliado o potencial da espectroscopia de infravermelho para estudar a qualidade bacteriana de alimentos. No capítulo 2, foi desenvolvido um modelo de calibração que permitiu prever com sucesso a concentração bacteriana de fiambre naturalmente contaminado, mantido em refrigeração durante 8 dias. Nesta parte, foi desenvolvida a metodologia que permitiu obter a melhor reprodutibilidade dos espectros das colónias de bactérias, com preparação mínima das amostras, que foi utilizada no trabalho subsequente. Foram realizadas várias tentativas para a obtenção de espectros de infravermelho, testando diferentes resoluções e número de scans. Os melhores resultados foram obtidos utilizando uma resolução espectral de 4 cm-1 e 32 varrimentos. De seguida, no capítulo 3, foi feita uma tentativa de identificar 22 bactérias provenientes de alimentos usando a espectroscopia de infravermelho associada a análise multivariada. A análise de componentes principais, utilizada como método exploratório, permitiu a formação de grupos distintos, cada um correspondendo a um género diferente, na grande maioria dos casos. Posteriormente, foi realizada uma análise hierárquica por clusters de forma a investigar a formação de grupos e a possibilidade de distinção de espécies dentro de um mesmo género de bactérias. Observou-se que a espectroscopia de infravermelho é adequada não só para a distinção de diferentes géneros, mas também para diferenciar espécies dentro de um mesmo género, com o uso simultâneo de análise de componentes principais e análise hierárquica por clusters. A utilização de espectroscopia de infravermelho e análise estatística multivariada foram também investigadas no capítulo 4 para confirmação da presença de Listeria monocytogenes e Salmonella spp., isoladas a partir de alimentos contaminados, após crescimento em meio selectivo. Isto permitiria a substituição dos métodos bioquímicos e serológicos que são usados para confirmar a presença destas bactérias patogénicas e que podem atrasar a obtenção de resultados por 2 dias. Os resultados obtidos permitiram a distinção de Salmonella spp. de outras bactérias que se possam confundir com elas. Por fim, no capítulo 5, o processamento por alta pressão, uma metodologia emergente que permite produzir alimentos microbiologicamente seguros e aumentar o seu tempo de prateleira, foi aplicada a 12 bactérias alimentares, de forma a determinar a sua resistência e os efeitos da pressão a nível das células. Foi aplicado um tratamento de 300 MPa, à temperatura ambiente e durante 15 minutos. As bactérias de Gram-negativo foram inativadas até níveis não detetáveis, enquanto as de Gram-positivo mostraram diferentes níveis de resistência. As espécies Bacillus cereus e Staphyloccus aureus decresceram apenas 2 unidades logarítmicas enquanto a espécie Listeria innocua diminuiu cerca de 5 unidades logarítmicas. A espectroscopia de infravermelho foi utilizada na análise das colónias bacterianas antes e após o tratamento por alta pressão, de forma a investigar as alterações que são provocadas nos componentes celulares com este tipo de processamento. Descobriu-se que a alta pressão altera bandas espectrais correspondentes a alguns componentes celulares, de entre os quais proteínas, lípidos, oligopolissacarídeos, grupos fosfato da parede celular e ácidos nucleicos, podendo indicar rutura da parede/membrana celular. Neste trabalho, a quantificação de bactérias e a sua classificação, bem como a análise de modificação nos componentes celulares após processamento por alta pressão foram realizados com sucesso. Assim, a espectroscopia de infravermelho demonstrou ser uma técnica bastante promissora para analisar bactérias provenientes de alimentos de uma forma simples e pouco dispendiosa